EXPOSURE OF BETA-H-CRYSTALLIN TO HYDROXYL RADICALS ENHANCES THE TRANSGLUTAMINASE-SUSCEPTIBILITY OF ITS EXISTING AMINE-DONOR AND AMINE-ACCEPTOR SITES

BetaH-crystallin was exposed to radiolytically generated hydroxyl radicals at defined radical concentrations. and its capacity to act as an amine-acceptor substrate and as an amine-donor substrate for transglutaminase were investigated. [C-14]Methylamine was used as a probe for labelling amine-acceptor sites: a novel biotinylated hexapeptide was used to label amine-donor sites. The results demonstrate that both primary amine incorporation and hexapeptide incorporation by transglutaminase are considerably increased after oxidative attack on the crystallin. The identity of the labelled subunits was established. and it is shown that, in both cases, this increased incorporation is not due to the production of new substrates, but that the existing incorporation sites become more susceptible. Moreover, using the newly developed probe, we could identify, for the first time, the major crystallin subunits active as amine-donor substrates (both before and after treatment) to be betaB1-, betaA3- and betaA4-crystallin. These data support the proposal that oxidative stress and transglutaminase activity may be jointly involved in the changes found in lens crystallins with age and in the development of cataract.